Quality over quantity: unraveling the contributions to cytoplasmic incompatibility caused by two coinfecting Cardinium symbionts

<p><i>Cardinium hertigii</i> is a common maternally-inherited bacterial endosymbiont of arthropods. Some <i>Cardinium</i> strains spread by increasing female fitness through the induction of cytoplasmic incompatibility (CI), which kills offspring of crosses between infected males and uninfected female hosts. CI is a two-step manipulation: first, sperm from male hosts are modified by the symbiont in a manner that causes offspring death. Second, when females are also infected, the symbiont reverses sperm modification in the egg cytoplasm, allowing offspring of infected females to survive and spread the symbiont. While <i>Cardinium</i> causes CI in many arthropod hosts, most of what is known about <i>Cardinium</i> CI stems from the symbiosis between the <i>Cardinium</i> strain <i>c</i>Eper1 and its minute parasitoid host, <i>Encarsia suzannae</i>. Here, we study a second <i>Cardinium</i> CI system in the wasp <i>Encarsia partenopea</i>. <i>Encarsia partenopea</i> harbors two <i>Cardinium</i> strains, <i>c</i>Eina2 and <i>c</i>Eina3, with the <i>c</i>Eina3 present at a much lower density than <i>c</i>Eina2. Using antibiotic treatments and crossing assays, we find that the low-density <i>c</i>Eina3 strain is responsible for CI, and that <i>c</i>Eina3 appears to modify sperm during the pupal stage, like the better known <i>c</i>Eper1. However, <i>c</i>Eina3 shows a markedly different localization pattern in male reproductive tissues. Instead of infecting sperm cells, <i>c</i>Eina3 is found in somatic cells at the base of the testis and around the seminal vesicle. This localization pattern suggests that <i>c</i>Eina3 may use a different sperm modification strategy from <i>c</i>Eper1, and highlights variation between these closely related symbioses.</p>